Abstract
Thermophilic enzymes are generally more thermally stable but are less active at moderate temperatures than are their mesophilic counterparts. Thermophilic enzymes with improved low-temperature activity that retain their high stability would serve as useful tools for industrial processes especially when robust biocatalysts are required. Here we show an effective way to explore amino acid substitutions that enhance the low-temperature catalytic activity of a thermophilic enzyme, based on a pairwise sequence comparison of thermophilic/mesophilic enzymes. One or a combination of amino acid(s) in 3-isopropylmalate dehydrogenase from the extreme thermophile Thermus thermophilus was/were substituted by a residue(s) found in the Escherichia coli enzyme at the same position(s). The best mutant, which contained three amino acid substitutions, showed a 17-fold higher specific activity at 25 °C compared to the original wild-type enzyme while retaining high thermal stability. The kinetic and thermodynamic parameters of the mutant showed similar patterns along the reaction coordinate to those of the mesophilic enzyme. We also analyzed the residues at the substitution sites from a structural and phylogenetic point of view.
Highlights
Enzymes have superior characteristics compared to inorganic catalysts that are often used in the chemical industry
Because our goal in this study was to establish a mesophilic level of low-temperature activity on TtIPMDH, we decided to compare the amino acid sequence of TtIPMDH with that of Escherichia coli IPMDH (EcIPMDH)
To search for amino acid substitutions that enhance the activity of TtIPMDHs at moderate temperature, one or more amino acids in TtIPMDH were substituted with residues found at the same positions in EcIPMDH according to the following three rules
Summary
Enzymes have superior characteristics compared to inorganic catalysts that are often used in the chemical industry. Psychrophilic organisms have low-temperature-adapted enzymes that are often less stable but catalytically more active at low temperatures compared with their mesophilic and thermophilic counterparts. TtIPMDH is a typical thermophilic enzyme that has high thermal stability and low activity at low temperatures[8]. Combinations of beneficial amino acid substitutions further improved the low-temperature activity, producing a mutant that displayed a catalytic activity two thirds of its mesophilic counterpart at 25 °C while retaining high thermal stability, only 2 °C lower than that of the T. thermophilus enzyme. We carried out a phylogenetic analysis of IPMDHs to find clues to establish guidelines to improve the catalytic activity of thermophilic enzymes at low temperature based on a pairwise sequence comparison of thermophilic/mesophilic enzymes
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have